应激和急性吗啡暴露对不同年龄大鼠海马CA1区突触可塑性及空间记忆的影响
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摘要
第一部分慢性应激和急性吗啡暴露对不同年龄大鼠海马CA1区突触可塑性的影响
目的:研究慢性平台应激和/或急性吗啡暴露对4周龄和10周龄Wistar大鼠海马CA1区突触可塑性的影响。
研究方法:
(1)试验分组:将4周龄雄性Wistar大鼠(51只)和10周龄雄性Wistar大鼠(51只)分别随机分为对照组,慢性应激组,吗啡组和慢性应激加吗啡组。每组又分为高频刺激(high frequencystimulation, HFS)组和低频刺激(Low frequency stimulation, LFS)组。
(2)应激模型:慢性平台应激。在一个光照良好的房子里,将要应激的大鼠放在一高1.6米的平台10cm×10cm上,每天9AM和3PM应激两次,每次30分钟,连续7天。四周龄慢性应激的大鼠是从第4周开始持续到第5周。10周龄慢性应激的大鼠是从第9周末开始持续到第10周末。
(3)LTP和LTD诱导、记录方法:将大鼠用戊巴比妥钠按每公斤体重40-60mg/kg腹腔注射麻醉后,在立体定向仪上定位电极,用电生理学标准来衡量电极的最佳深度和试验后的尸体脑部检查来检验定位正确与否。试验时刺激Schaffer侧支/共同通路从海马CA1区记录到场电位fEPSP (field excitatory synaptic potentials, fEPSP)的。记录时先稳定基线40分钟,然后给予高频刺激HFS诱导LTP(long-term potentiation, LTP)或低频刺激LFS诱导LTD(long-term depression, LTD)。慢性应激加吗啡组和吗啡组的大鼠在记录基线40分钟后给予吗啡3mg/kg腹腔注射,90分钟后诱导LTP或LTD。HFS是由10串刺激组成,每串之间间隔2s,每串20个刺激,每个刺激之间间隔5ms (200HZ)。LFS是由900个脉冲组成(1HZ)。
(4)统计学方法:采用t检验。
结果:(1)对照组10周龄LTP的EPSP幅度明显比4周龄要大(P<0.05),10周龄和4周龄大鼠慢性应激后的LTP与对照组相比明显小(P<0.05);10周龄大鼠在慢性应激后不能诱导出LTD,而慢性
Part I The Effect of Chronic Stress and Acute Morphine Exposure onSynaptic Plasticity of Hippocampal CA1 Area in Rats of Different Age inVivo
Objectives: To study the effects of chronic stress and acutemorphine exposure on synaptic plasticity of hippocampal CA1 Area in4-or10-week-old rats.
Methods: Fifty-one 4-week-old and 51 10-week-old male Wistarrats were randomly assigned to control group, chronic stress group,morphine group, chronic stress and morphine group, respectively. Eachgroup was then randomly assigned to high frequency stimulation (HFS)group and low frequency stimulation (LFS) group. Behavioral stress wasevoked by placing the rats on an elevated platform (EP, 10cm×10cm,high 1.6m) in the middle of a brightly lit room for 30min per time. Ratsreceiving chronic EP stress were placed on the EP twice per day at 9AMand 3PM for 7days (EP stress for 4-week-old rats was from 4th week to5th week;for 10-week-old rats from ninth week to 10th week).
Experiments were carried out under pentobarbitone sodium (40-/60mg/kg, i.p) anesthesia. Electrode implantation sites were identified byusing stereotaxic coordinates. Recordings of field excitatory postsynapticpotential (fEPSP) were made from the CA1 stratum radiatum of thehippocampus in response to ipsilateral stimulation of the Schaffercollateral/commissural pathway similar to those described. The optimaldepth of the wire electrodes in the stratum radiatum of the CA1 area ofthe dorsal hippocampus was determined by using electrophysiologicalcriteria and was verified by post mortem examination. After 40 minutesstable baseline recording, HFS (200HZ) or LFS (1HZ) was given toinduce LTP or LTD, respectively. For the rats of chronic stress andmorphine group and morphine group, morphine (3mg/kg, i.p) was givenafter 40 minutes stable baseline recording,then HFS or LFS was given toinduce LTP or LTD after 90 minutes recording. Low frequencystimulation (LFS) consisted of 900 pulses at 1Hz. High frequency
stimulation (HFS) consisted of 10 trains of 20 stimuli, inter-stimulusinterval 5 ms (200HZ), inter-train interval 2 s.Statistical comparisons were made using Student's t-test.Results: (1) The EPSP amplitude of LTP in 10-week-old rats withoutchronic EP stress was larger than that in 4-week-old rats (P<0.05). LTPwas impaired after chronic stress either in 10-week-old rats comparedwith control (P<0.05) or in 4-week-old rats compared with control(P<0.05). LTD was facilitated in 4-week-old stressed rats compared withcontrol (P<0.05), However low-frequency stimulation failed to induceLTD in 10-week-old stressed rats.(2) Acute morphine exposure (3mg/kg, i.p) facilitated the induction ofLTP in 10-week-old animals compared with control (P<0.05) andimpaired the induction of LTP in 4-week-old animals compared withcontrol (P<0.05);LTD was facilitated after acute morphine exposureeither in 10-week-old rats compared with control (P<0.05) or in4-week-old rats compared with control (P<0.05).(3) LTP was facilitated by acute morphine exposure (3mg/kg, i.p) in4-week-old stressed rats compared with control (P<0.05) and impaired in10-week-old stressed rats compared with control (P<0.05).Low-frequency stimulation (LFS) failed to induce LTD in 10-week-oldstressed rats but stable LTD was stilled induced and facilitated in4-week-old stressed rats after acute morphine exposure compared withcontrol (P<0.05).Conclusion: (1) chronic stress and morphine had different effects onLTP and LTD in 4-or10-week-old rats and it showed significantdifference between two ages. LTP and LTD were facilitated in chronicstress and morphine group of 4-week-old rats;LTP was impaired andLTD was completely inhibited in the same group of 10-week-old rats. Theresults also showed the difference between two ages.(2) The effect of acute morphine exposure on synaptic plasticity (LTPand LTD) after chronic stress was changed, either in 10-week-old rats orin 4-week-old rats.
Part II Effects of Stress and Acute Morphine Exposure on SpatialMemory in Rats of Different AgeObjectives: To study the effects of acute and chronic stress andacute morphine exposure (2mg/kg) on spatial memory in 4-or10-week-old rats.Methods: 4-week-old Male Wistar rats (49) and 10-week-old MaleWistar rats (52) were randomly assigned to saline group (control group),chronic stress and saline group (chronic stress group), acute stress andsaline group (acute stress), chronic stress and morphine group, acutestress and morphine group and morphine group.Stress protocol and morphine injection: Behavioral stress wasevoked by placing the rats on an elevated platform (EP) in the middle of abrightly lit room for 30min per time. Rats receiving chronic EP stresswere placed on the EP twice per day at 9AM and 3PM,lasting 7days (EPstress for 4-week-old rats was from 4th week to 5th week;for10-week-old rats from ninth week to 10th week). Rats for chronic stressstill received EP stress one time before training and probe trial. Acutestress group received EP stress one time only before training and probetrial. Morphine was injected peritoneally (i.p) one time before probe trialat the dose of 2mg/kg, so did the same for saline injection.Training procedure: all the experiments were practiced in the Morriswater maze. Before training, a 180 s free swim trial was run in which theplatform was removed so that animals were adapted to temperature andenvironment of Morris water maze. During the training, a submergedPerspex platform (14cm×14cm) was placed 62.5cm away from the edgeof Morris water maze Which make animal to learn the location of theplatform that could be used to escape from swimming. The trainingsession consisted of eight trials (maximum trial duration=180s) with eight
different starting positions that were equally distributed around theperimeter of the maze. Total 16 trials were given as eight sessions with30min inter trial interval per day for consecutive two days.Probe trial: Thirty minutes after the end of the training session onday 2, a probe trial was given which consisted of a 180s free swim periodwithout the platform. Time spent in the Platform quadrant (targetquadrant) was calculated. It was given 30minute after the final trainingtrial. Swim path for all training and probe trial was monitored using anautomatic tracking system.Statistical comparison was made by using t test or one-way ANOVAfollowed by LSD (SPSS 10.0).Result(1) The effect of stress and acute morphine exposure on the spatialmemory retention in 4-week-old ratsRats of acute stress group, acute stress and morphine group andmorphine group spent significantly less time in the target quadrant thanthat of control group, respectively (P<0.05). Time in the target quadrantdid not differ in chronic stress group, chronic stress and morphine groupcompared with control (P>0.05).(2) The effect of stress and acute morphine exposure on the spatialmemory retention in 10-week-old ratsRats of chronic stress group and chronic stress and morphine groupspent significantly more time in the target quadrant than that of controlgroup (P<0.05);Rats of acute stress group spent significantly less time inthe target quadrant than that of control group (P<0.05). Time in the targetquadrant did not differ in acute stress and morphine group, morphinegroup compared with control group (P>0.05).(3) Comparison of the effect of stress and acute morphine exposure on thespatial memory retention between 4-week-old rats and 10-week-od rats.10-week-old rats in chronic stress group, chronic stress andmorphine group, morphine group, acute stress and morphine group spent
significantly more time in the target quadrant than that of 4-week-old ratsin the same groups, respectively (P<0.05).Time in the target quadrant didnot differ in acute stress group and control group between 4-week-old ratsand 10-week-old rats (P>0.05).(4) Comparison of speed of groups in 4-or 10-week-old rats: Swimmingspeed did not differ in all groups of 4-or 10-week-old rats (P>0.05).Conclusion: (1) Acute EP stress induced impairment of memoryretention in 4-or10-week-old rats, respectively,but it did not differbetween 4-week-old rats and 10-week-old rats. Chronic EP stress inducedenhancement of memory retention in 10-week-old rats and had no effecton memory retrieval in 4-week-old rats. It showed difference betweentwo age groups.(2) Morphine at the dose of 2mg/kg induced impairment of memoryretention in 4-week-old rats and had no effect on memory retention in10-week-old rats. It showed difference between two age groups.(3) Acute EP stress and morphine induced impairment of memoryretention in 4-week-old rats and had no effect on memory in 10-week-oldrats. It showed difference between two age groups. Chronic stress andmorphine had no effect on memory retention in 4-week-old rats andinduced enhancement of memory retention in 10-week-old rats. It alsoshowed difference between two different ages.
目的:研究慢性平台应激和/或急性吗啡暴露对4周龄和10周龄Wistar大鼠海马CA1区突触可塑性的影响。
研究方法:
(1)试验分组:将4周龄雄性Wistar大鼠(51只)和10周龄雄性Wistar大鼠(51只)分别随机分为对照组,慢性应激组,吗啡组和慢性应激加吗啡组。每组又分为高频刺激(high frequencystimulation, HFS)组和低频刺激(Low frequency stimulation, LFS)组。
(2)应激模型:慢性平台应激。在一个光照良好的房子里,将要应激的大鼠放在一高1.6米的平台10cm×10cm上,每天9AM和3PM应激两次,每次30分钟,连续7天。四周龄慢性应激的大鼠是从第4周开始持续到第5周。10周龄慢性应激的大鼠是从第9周末开始持续到第10周末。
(3)LTP和LTD诱导、记录方法:将大鼠用戊巴比妥钠按每公斤体重40-60mg/kg腹腔注射麻醉后,在立体定向仪上定位电极,用电生理学标准来衡量电极的最佳深度和试验后的尸体脑部检查来检验定位正确与否。试验时刺激Schaffer侧支/共同通路从海马CA1区记录到场电位fEPSP (field excitatory synaptic potentials, fEPSP)的。记录时先稳定基线40分钟,然后给予高频刺激HFS诱导LTP(long-term potentiation, LTP)或低频刺激LFS诱导LTD(long-term depression, LTD)。慢性应激加吗啡组和吗啡组的大鼠在记录基线40分钟后给予吗啡3mg/kg腹腔注射,90分钟后诱导LTP或LTD。HFS是由10串刺激组成,每串之间间隔2s,每串20个刺激,每个刺激之间间隔5ms (200HZ)。LFS是由900个脉冲组成(1HZ)。
(4)统计学方法:采用t检验。
结果:(1)对照组10周龄LTP的EPSP幅度明显比4周龄要大(P<0.05),10周龄和4周龄大鼠慢性应激后的LTP与对照组相比明显小(P<0.05);10周龄大鼠在慢性应激后不能诱导出LTD,而慢性
Part I The Effect of Chronic Stress and Acute Morphine Exposure onSynaptic Plasticity of Hippocampal CA1 Area in Rats of Different Age inVivo
Objectives: To study the effects of chronic stress and acutemorphine exposure on synaptic plasticity of hippocampal CA1 Area in4-or10-week-old rats.
Methods: Fifty-one 4-week-old and 51 10-week-old male Wistarrats were randomly assigned to control group, chronic stress group,morphine group, chronic stress and morphine group, respectively. Eachgroup was then randomly assigned to high frequency stimulation (HFS)group and low frequency stimulation (LFS) group. Behavioral stress wasevoked by placing the rats on an elevated platform (EP, 10cm×10cm,high 1.6m) in the middle of a brightly lit room for 30min per time. Ratsreceiving chronic EP stress were placed on the EP twice per day at 9AMand 3PM for 7days (EP stress for 4-week-old rats was from 4th week to5th week;for 10-week-old rats from ninth week to 10th week).
Experiments were carried out under pentobarbitone sodium (40-/60mg/kg, i.p) anesthesia. Electrode implantation sites were identified byusing stereotaxic coordinates. Recordings of field excitatory postsynapticpotential (fEPSP) were made from the CA1 stratum radiatum of thehippocampus in response to ipsilateral stimulation of the Schaffercollateral/commissural pathway similar to those described. The optimaldepth of the wire electrodes in the stratum radiatum of the CA1 area ofthe dorsal hippocampus was determined by using electrophysiologicalcriteria and was verified by post mortem examination. After 40 minutesstable baseline recording, HFS (200HZ) or LFS (1HZ) was given toinduce LTP or LTD, respectively. For the rats of chronic stress andmorphine group and morphine group, morphine (3mg/kg, i.p) was givenafter 40 minutes stable baseline recording,then HFS or LFS was given toinduce LTP or LTD after 90 minutes recording. Low frequencystimulation (LFS) consisted of 900 pulses at 1Hz. High frequency
stimulation (HFS) consisted of 10 trains of 20 stimuli, inter-stimulusinterval 5 ms (200HZ), inter-train interval 2 s.Statistical comparisons were made using Student's t-test.Results: (1) The EPSP amplitude of LTP in 10-week-old rats withoutchronic EP stress was larger than that in 4-week-old rats (P<0.05). LTPwas impaired after chronic stress either in 10-week-old rats comparedwith control (P<0.05) or in 4-week-old rats compared with control(P<0.05). LTD was facilitated in 4-week-old stressed rats compared withcontrol (P<0.05), However low-frequency stimulation failed to induceLTD in 10-week-old stressed rats.(2) Acute morphine exposure (3mg/kg, i.p) facilitated the induction ofLTP in 10-week-old animals compared with control (P<0.05) andimpaired the induction of LTP in 4-week-old animals compared withcontrol (P<0.05);LTD was facilitated after acute morphine exposureeither in 10-week-old rats compared with control (P<0.05) or in4-week-old rats compared with control (P<0.05).(3) LTP was facilitated by acute morphine exposure (3mg/kg, i.p) in4-week-old stressed rats compared with control (P<0.05) and impaired in10-week-old stressed rats compared with control (P<0.05).Low-frequency stimulation (LFS) failed to induce LTD in 10-week-oldstressed rats but stable LTD was stilled induced and facilitated in4-week-old stressed rats after acute morphine exposure compared withcontrol (P<0.05).Conclusion: (1) chronic stress and morphine had different effects onLTP and LTD in 4-or10-week-old rats and it showed significantdifference between two ages. LTP and LTD were facilitated in chronicstress and morphine group of 4-week-old rats;LTP was impaired andLTD was completely inhibited in the same group of 10-week-old rats. Theresults also showed the difference between two ages.(2) The effect of acute morphine exposure on synaptic plasticity (LTPand LTD) after chronic stress was changed, either in 10-week-old rats orin 4-week-old rats.
Part II Effects of Stress and Acute Morphine Exposure on SpatialMemory in Rats of Different AgeObjectives: To study the effects of acute and chronic stress andacute morphine exposure (2mg/kg) on spatial memory in 4-or10-week-old rats.Methods: 4-week-old Male Wistar rats (49) and 10-week-old MaleWistar rats (52) were randomly assigned to saline group (control group),chronic stress and saline group (chronic stress group), acute stress andsaline group (acute stress), chronic stress and morphine group, acutestress and morphine group and morphine group.Stress protocol and morphine injection: Behavioral stress wasevoked by placing the rats on an elevated platform (EP) in the middle of abrightly lit room for 30min per time. Rats receiving chronic EP stresswere placed on the EP twice per day at 9AM and 3PM,lasting 7days (EPstress for 4-week-old rats was from 4th week to 5th week;for10-week-old rats from ninth week to 10th week). Rats for chronic stressstill received EP stress one time before training and probe trial. Acutestress group received EP stress one time only before training and probetrial. Morphine was injected peritoneally (i.p) one time before probe trialat the dose of 2mg/kg, so did the same for saline injection.Training procedure: all the experiments were practiced in the Morriswater maze. Before training, a 180 s free swim trial was run in which theplatform was removed so that animals were adapted to temperature andenvironment of Morris water maze. During the training, a submergedPerspex platform (14cm×14cm) was placed 62.5cm away from the edgeof Morris water maze Which make animal to learn the location of theplatform that could be used to escape from swimming. The trainingsession consisted of eight trials (maximum trial duration=180s) with eight
different starting positions that were equally distributed around theperimeter of the maze. Total 16 trials were given as eight sessions with30min inter trial interval per day for consecutive two days.Probe trial: Thirty minutes after the end of the training session onday 2, a probe trial was given which consisted of a 180s free swim periodwithout the platform. Time spent in the Platform quadrant (targetquadrant) was calculated. It was given 30minute after the final trainingtrial. Swim path for all training and probe trial was monitored using anautomatic tracking system.Statistical comparison was made by using t test or one-way ANOVAfollowed by LSD (SPSS 10.0).Result(1) The effect of stress and acute morphine exposure on the spatialmemory retention in 4-week-old ratsRats of acute stress group, acute stress and morphine group andmorphine group spent significantly less time in the target quadrant thanthat of control group, respectively (P<0.05). Time in the target quadrantdid not differ in chronic stress group, chronic stress and morphine groupcompared with control (P>0.05).(2) The effect of stress and acute morphine exposure on the spatialmemory retention in 10-week-old ratsRats of chronic stress group and chronic stress and morphine groupspent significantly more time in the target quadrant than that of controlgroup (P<0.05);Rats of acute stress group spent significantly less time inthe target quadrant than that of control group (P<0.05). Time in the targetquadrant did not differ in acute stress and morphine group, morphinegroup compared with control group (P>0.05).(3) Comparison of the effect of stress and acute morphine exposure on thespatial memory retention between 4-week-old rats and 10-week-od rats.10-week-old rats in chronic stress group, chronic stress andmorphine group, morphine group, acute stress and morphine group spent
significantly more time in the target quadrant than that of 4-week-old ratsin the same groups, respectively (P<0.05).Time in the target quadrant didnot differ in acute stress group and control group between 4-week-old ratsand 10-week-old rats (P>0.05).(4) Comparison of speed of groups in 4-or 10-week-old rats: Swimmingspeed did not differ in all groups of 4-or 10-week-old rats (P>0.05).Conclusion: (1) Acute EP stress induced impairment of memoryretention in 4-or10-week-old rats, respectively,but it did not differbetween 4-week-old rats and 10-week-old rats. Chronic EP stress inducedenhancement of memory retention in 10-week-old rats and had no effecton memory retrieval in 4-week-old rats. It showed difference betweentwo age groups.(2) Morphine at the dose of 2mg/kg induced impairment of memoryretention in 4-week-old rats and had no effect on memory retention in10-week-old rats. It showed difference between two age groups.(3) Acute EP stress and morphine induced impairment of memoryretention in 4-week-old rats and had no effect on memory in 10-week-oldrats. It showed difference between two age groups. Chronic stress andmorphine had no effect on memory retention in 4-week-old rats andinduced enhancement of memory retention in 10-week-old rats. It alsoshowed difference between two different ages.
引文
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